Summary

CD105, a marker of endothelial cells, is abundantly expressed in tissues
undergoing angiogenesis and is a receptor for transforming growth
factorβ. The pivotal role of CD105 in the vascular system was
demonstrated by the severe vascular defects that occur in CD105-knockout mice,
but the exact mechanisms for CD105 regulation of vascular development have not
been fully elucidated. In light of the function of CD105 and the importance of
hypoxia in neovascularisation, we speculated that CD105 is involved in
hypoxia-initiated angiogenesis. Using tissue-cultured human microvascular
endothelial cells, we have investigated the effects of hypoxic stress on CD105
gene expression. Hypoxia induced a significant increase in membrane-bound and
secreted CD105 protein levels. CD105 mRNA and promoter activity were also
markedly elevated, the latter returning to the basal level after 16 hours of
hypoxic stress. Hypoxia induced cell cycle arrest at the G0/G1 phases and
massive cell apoptosis after 24 hours through a reduction in the Bcl-2 to Bax
ratio, downregulation of Bcl-XL and Mcl-1, and upregulation of
caspase-3 and caspase-8. The consequence of CD105 upregulation was revealed
using an antisense approach and a TUNEL assay. Suppression of CD105 increased
cell apoptosis under hypoxic stress in the absence of TGFβ1. Furthermore,
hypoxia and TGFβ1 synergistically induced apoptosis in the
CD105-deficient cells but not in the control cells. We conclude that hypoxia
is a potent stimulus for CD105 gene expression in vascular endothelial cells,
which in turn attenuates cell apoptosis and thus contributes to
angiogenesis.

Considering the pro-angiogenic function of CD105, we speculated that there
might be an association between oxygen deprivation and CD105 gene expression.
Should such a relationship exist, it would not only explain the augmented
expression of CD105 in tumours and ischaemic tissues but would also have
therapeutic implications for many angiogenic diseases. Furthermore,
elucidating the function of CD105 under hypoxic stress would improve our
understanding of the pathogenesis of several vascular diseases in which CD105
is implicated.

Materials and Methods

Cell culture

Human dermal microvascular endothelial cells (HDMECs) (Clonetics, San
Diego, CA) were grown in complete growth medium composed of MCDB 131, 10 ng/ml
epithelial growth factor, 10% (v/v) foetal calf serum (Invitrogen), 2 mM
glutamine plus 100 μg/ml penicillin and 100 μg/ml streptomycin. For
hypoxic exposure, confluent HDMECs were incubated within a modular incubator
chamber filled with a gas mixture of 0.2% oxygen, 5% carbon dioxide and 94.8%
nitrogen placed in a 37°C incubator. Control cells with the same
confluency were maintained at normoxic conditions (20% oxygen, 5% carbon
dioxide and 75% nitrogen) in the same incubator. To study cell apoptosis under
hypoxic stress, HDMECs in complete growth medium were cultured in the hypoxic
chamber in the presence or absence of recombinant human TGFβ1 (R&D
Systems, Abingdon, UK) for 24 hours. We used ELISAs that were sensitive for
TGFβ1 and TGFβ3 [and can detect as little as 20 pg/ml of TGFβ1
and 100 pg/ml of TGFß3 (Li et al.,
2000b)], but we did not detect TGFβ1 or TGFβ3 in the
complete growth medium. However, in order to eliminate any trace amount of
TGFβ1 and TGFβ3 in the medium, neutralising antibodies to TGFβ1
and TGFβ3 were administered in parallel experiments. Ten μg/ml of
chick anti-TGFβ1 (R&D Systems) and an equal amount of goat
anti-TGFβ3 (R&D Systems) were added to the cultures in the absence of
exogenously added TGFβ1 or TGFβ3 during exposure to hypoxia. This
antibody concentration was sufficient to neutralise the bioactivity of 2,000
pg/ml of TGFβ1 and 250 pg/ml of TGFβ3.

To suppress CD105 expression in HDMECs, an antisense approach was applied
as described previously (Li et al.,
2000a). Briefly, the 16-mer antisense phosphorothioate-modified
oligodeoxynucleotide (AS ODN: 5′-ATGCTGTCCACGTGGG-3′) and the
scrambled control ODN with the same base composition (SC ODN:
5′-ACTCGTGC-TACGGTGG-3′) were synthesised (Applied Biosystems,
Foster City, CA). Subconfluent HDMECs grown in 35 mm petri dishes were
transfected with 0.5 nM/ml of AS or SC ODN plus 8 μg/ml of DMRIE-C
(Invitrogen) in serum-free medium. After overnight incubation, the medium was
replenished with complete growth medium, and incubation was continued for an
additional 48 hours in normoxia. Thereafter the medium was replaced with fresh
complete growth medium, and the cultures were exposed to hypoxia for up to 24
hours.

Analysis of CD105 protein expression

Cell surface expression of CD105 protein in AS-, SC-treated or untreated
cells was quantified by flow cytometry as described previously
(Li et al., 2000a). Briefly,
105 cells per tube were incubated with 50 μl (10 μg/ml in
PBS) of anti-CD105 mAb E9 or pre-immunised mouse serum as a negative control
antibody (10 μg/ml in PBS) on ice for 1 hour and washed twice with cold
PBS. mAb 44G4 to human CD105 (Gougos and
Letarte, 1988) was also employed for comparison with mAb E9. After
incubation with a fluorescein-labelled
F(ab́)2 fragment of rabbit
anti-mouse antibody (1/40; DAKO, Denmark) for 30 minutes on ice, the cells
were washed and resuspended in 0.3 ml of 2% buffered formalin and analysed on
a Becton Dickinson FACScan flow cytometer.

Analysis of CD105 mRNA by northern blotting

Total RNA from HDMECs was extracted using guanidinium
thiocyanate/phenol/chloroform (Chomczynski
and Sacchi, 1987). RNA samples (15 μg) were denatured and
fractionated in 1% (w/v) agarose/2.8% (v/v) formaldehyde gel and blotted onto
nitrocellulose. The CD105 probe used was a 2.3 kb fragment excised with
EcoRI from the pcEXV-EndoL plasmid
(Lastres et al., 1996) and
labelled with 32P by random prime labelling. The blot was
hybridised with the 32P-labelled probe and the hybridisation was
revealed by a phosphorimager (Molecular Dynamics), then analysed using Image
Quant software. The blots were rehybridised with 32P-labelled probe
for GAPDH for use as a loading control.

Luciferase reporter gene assay

To determine the level of CD105 promoter activity, plasmid pXP2 harbouring
the 2.8 kb (-2450/+350) CD105 promoter
(Rius et al., 1998) and a
downstream firefly luciferase gene was used for transient transfection of
HDMECs. Internal normalisation was performed by cotransfection of the pXP2
plasmid with CMVβgal, a β-galactosidase expression vector driven by
the cytomegalovirus (CMV) promoter. Transfection of HDMECs was carried out
using the liposome-mediated gene transfer technique. Briefly, cells were
seeded at 1×105 cells per 35 mm dish and the following day
were transfected with 8 μg/ml of DMRIE-C (Invitrogen) plus 1 μg of
plasmid CMVβgal mixed with 2 μg of pXP2 in serum-free medium.
Twenty-four hours after transfection, the cultures were replenished with
complete medium and placed in hypoxic or normoxic conditions for the
designated time periods. Thereafter, the cells were harvested and the
enzymatic activity determined. Luciferase and β-galactosidase activities
were measured on a TD-20/20 Luminometer (Promega, WI) and a colorimetric plate
reader (Labsystems), respectively, using kits from Promega (UK).

Cell cycle analysis

Cell cycle distribution was evaluated by propidium iodide staining of
nuclei and flow cytometric analysis (Li et
al., 1997). 1×106 cells were fixed with 2 ml cold
70% (v/v) ethanol in PBS, immediately mixed and slowly agitated for 15 minutes
at room temperature. After two washes with PBS, 0.7 ml of 0.2 mg/ml pepsin
(Sigma) in 2 M HCl was added to the pelleted cells for simultaneous
proteolysis and DNA denaturation at 37°C for 30 minutes. The hydrolysis
was terminated by addition of 2 ml of 1 M Tris (pH 10). Cells were washed
twice and incubated in 0.3 ml PBS containing 10 μg/ml propidium iodide for
15 minutes on ice. To determine the proportion of cells in various phases of
the cell cycle, propidium iodide staining of the DNA in 2×104
nuclei was quantified using a Becton Dickinson FACScan flow cytometer.

Analysis of apoptosis

The terminal deoxynucleotidyltransferase-mediated dUTP end labelling
(TUNEL) assay was used to identify cell apoptosis. Cells were seeded onto
chamber slides (Nunc) at a density of 2×104 cells per 0.5 ml
per well, grown overnight and subjected to hypoxic or normoxic conditions.
Fragmented DNA staining of apoptotic cells was carried out using a commercial
kit (Roche, Mannheim, Germany). Briefly, the cells were rinsed twice with
pre-warmed PBS followed by fixation using 4% paraformaldehyde for 1 hour at
room temperature. The fixed cells were permeabilised by incubation with 0.1%
Triton X-100 in 0.1% sodium citrate for 2 minutes. The cells were rinsed again
with PBS and incubated with 50 μl per sample of TUNEL reaction mixture for
1 hour at 37°C. A negative control was included in each staining wherein
only the labelling solution was added.

TUNEL staining of cells for FACS analysis was carried out using the same
kit as described above. The cells were fixed with a freshly prepared
paraformaldehyde solution (4% in PBS, pH 7.4) for 1 hour at room temperature.
After one wash with PBS, cells were permeabilised using 0.1% Triton X-100 in
0.1% sodium citrate for 2 minutes on ice, followed by two washes with PBS. The
permeabilised cells were stained with 50 μl/sample of the TUNEL reaction
mixture containing the enzyme or the labelling solution without the enzyme as
a negative control and incubated for 1 hour at 37°C in a humidified
chamber in the dark. Finally, the cells were washed twice with PBS and
resuspended in 0.5 ml PBS for FACS analysis. For quantification of anti- and
pro-apoptotic proteins, cells were harvested, fixed and permeabilised as
described above. Antibodies to Bcl-2, Bcl-XL, Bax, Mcl-1, caspase-3
and caspase-8 (BD Biosciences) were incubated with 5×105
cells/tube for 1 hour on ice. Pre-immunised rabbit serum or isotype matched
mouse immunoglobulins were used as negative controls. The FITC-conjugated
secondary antibodies (DAKO) were added and incubated for 30 minutes on ice.
Flow cytometric analysis was carried out on a FACScan.

Results

Hypoxia upregulates CD105 protein levels

HDMECs collected from normoxic or hypoxic cultures were stained with mAb
E9, which reacts specifically with human CD105
(Pichuantes et al., 1997), and
cell surface CD105 expression was quantified by flow cytometry. CD105 levels
were markedly elevated under hypoxic conditions compared with normoxic
conditions, especially after 16 hours (Fig.
1A). The geometric mean fluorescence intensity increased from
149.5±13.5 in normoxic conditions to 278.0±19.0 after 16 hours
of hypoxic culture, that is, an 86.3% increase in cell surface CD105. FACS
analysis was used to compare the reactivity of mAbs E9 and 44G4 against human
CD105 on the same HDMECs (Gougos and
Letarte, 1988; Pichuantes et
al., 1997). Both mAbs reacted with ∼100% of the confluent
cells in normoxic conditions. mAbs 44G4 and E9 bound equally well to HDMECs,
resulting in almost identical results under hypoxic conditions (data not
shown). Next, analysis of total CD105 present in cellular extracts was carried
out by immunoblotting analysis. Cell extracts were prepared from the same
batch of HDMECs used for flow cytometry analysis and subjected to
electrophoresis under non-reducing conditions, blotted to PVDF membrane and
probed using a mAb against CD105 and a goat anti-α-actin antibody. In
contrast to flow cytometry analysis, which detects only membrane-bound
antigen, immunoblotting identifies total CD105. As depicted in
Fig. 1B, two specific bands
with molecular weights of approximately 180 and 95 kDa were observed. The 180
kDa band corresponds to the mature dimeric form of CD105 and showed a∼
threefold greater intensity after 16 hours of hypoxic culture compared
with normoxic controls. The smaller band is likely to be a monomer of CD105
(Paquet et al., 2001). Both
the dimer and the monomer forms of CD105 were markedly increased under hypoxic
conditions and were maximal at 16 hours, indicating that hypoxia significantly
elevated the levels of total cellular CD105 protein. When the blots were
stripped and reprobed using mAb 44G4, the same bands were observed, with
similar intensity, confirming that the two mAbs to human CD105 had almost
equal reactivity.

Hypoxia induces CD105 protein expression. HDMECs were cultured under
hypoxic conditions for up to 24 hours, and CD105 expression was quantified by
flow cytometry (A), immunoblotting (B) and ELISA (C) as described in Materials
and Methods. (A) Hypoxic culture resulted in a maximal cell surface
expression of CD105 at 16 hours (86.3% increase). The data represent five
samples at each time point collected from five separate experiments
(*P<0.05 and **P<0.01 compared with 0 hours as
analysed by one-way ANOVA followed by the Duncan test). (B) Cell
extracts were resolved on 4-7.5% SDS-PAGE under non-reducing conditions and
electrophoretically transferred onto membranes. The blot was probed using mAb
E9 and a rabbit anti-α-actin antibody. The maximal expression (as
quantified on a densitometer) of CD105 was seen at 16 hours of hypoxic
culture. Similar results were observed when the blots were reprobed using mAb
44G4 (data not shown). The bar chart shows the CD105 signal intensity relative
to actin, pooled from three experiments. (C) Conditioned medium was
collected, and soluble CD105 levels were quantified using a chemiluminescence
ELISA system. CD105 levels peaked at 16 hours of hypoxic culture. The data
represent six replicates at each time point pooled from three separate
experiments (*P<0.05 and **P<0.01 compared with 0
hours as analyzed by one-way ANOVA followed by the Duncan test). Vertical bars
indicate the standard error of the means.

CD105 levels in the blood of patients with breast cancer and those with
atherosclerosis are elevated and associated with disease progression. Thus
CD105 could be a marker for certain clinical conditions
(Blann et al., 1996;
Li et al., 2000c). In the
present study, CD105 levels were quantified in conditioned medium collected
from cells used for protein and mRNA analysis. The levels of secreted CD105
were increased after 1 hour of oxygen deprivation, peaked at 16 hours in
hypoxic cultures and remained higher than controls at 24 hours
(Fig. 1C). However the
biological implications of the increase in CD105 are unknown. The profile of
secreted CD105 in the oxygen-deprived medium was comparable to cell surface
CD105 observed by flow cytometry and total CD105 estimated by immunoblotting
analysis.

Hypoxia upregulates CD105 mRNA levels and promoter activity

To analyse whether the levels of CD105 transcripts were also affected by
hypoxia, total RNA was fractionated and hybridised with radiolabelled CD105
and GAPDH cDNAs, visualised on a phosphorimager and quantified using a
densitometer. Hypoxia elevated CD105 mRNA levels, which was evident even after
1 hour, compared with normoxic culture
(Fig. 2A). The highest
expression of CD105 mRNA (a ∼threefold increase) occurred at 3 hours. The
relatively weak signal for CD105 mRNA after 24 hours of hypoxic culture was
considered to reflect hypoxia-induced cell cycle arrest and apoptosis, which
was confirmed by propidium iodide staining and a TUNEL assay, as described
below. These low levels of CD105 mRNA were concomitant with high expression
levels of CD105 protein (Fig.
1), in agreement with the high stability of the protein
(Paquet et al., 2001). To
assess whether the increased CD105 expression in hypoxic conditions was due to
an increased promoter activity, CD105 promoter activity was examined using a
luciferase reporter assay. CD105 promoter activity was significantly induced
by hypoxia, reaching a maximal level after 3-6 hours of hypoxic culture
(Fig. 2B). Longer durations of
hypoxia (more than 16 hours) led to basal levels of the promoter activity,
probably owing to cell cycle arrest and apoptosis.

Effect of hypoxia on CD105 transcription. (A) Northern blot analysis. Total
RNA was extracted from HDMECs and fractionated on a 1% denaturing agarose gel.
After blotting onto a nitrocellulose membrane, the fractionated RNA was probed
using 32P-labelled cDNAs for CD105 or GAPDH, visualised on a
phosphorimager and quantified using a densitometer. Maximal expression of
CD105 mRNA was observed at 3 hours with a ∼threefold increase over
normoxic culture. The bar chart represents CD105 mRNA relative to GAPDH mRNA
signal intensity collected from three experiments. (B) Hypoxia activates the
CD105 promoter. HDMECs (the same batch of cells as for panel A) co-transfected
with plasmid pXP2/pCD105/luc and CMVβgal were grown under hypoxic
condition for up to 24 hours. Luciferase activity was determined and
normalised to β-galactosidase activity. CD105 promoter activity peaked
between 3 hours and 6 hours of culture (*P<0.05 and
**P<0.01 compared with 0 hours as analysed by one-way ANOVA
followed by the Duncan test). Data represent six replicates at each time point
collected from three separate experiments. Vertical bars indicate standard
error of the means.

Hypoxia induces cell cycle arrest and apoptosis in HDMECs

Since persistent hypoxia induces alterations in cell cycle and apoptosis in
certain cell types (Carmeliet et al.,
1998), an analysis of cell cycle and apoptosis was performed in
hypoxic HDMECs. Prolonged exposure to hypoxia (24 hours) led to a dramatic
alteration in cell cycle, that is, more cells arrested at the G0/G1 phases and
fewer cells undergoing DNA synthesis (S phase). Moreover, DNA fragmentation
was observed in a considerable proportion of cells (approximately 21% of the
total population – blue profile) after 24 hours of hypoxic culture,
implying massive cell apoptosis (Fig.
3A). A TUNEL assay was carried out in parallel on the same batch
of cells to identify cells with fragmented DNA. Under normoxic conditions, few
apoptotic cells were observed. In contrast, in cells grown under hypoxic
conditions DNA fragmentation occurred in a time-dependent manner: prolonged
exposure to oxygen deprivation induced substantial cell apoptosis
(Fig. 3B). The highest
proportion of apoptotic cells was detected after 24 hours of hypoxic culture,
which is consistent with the data obtained by propidium iodide staining.

Cell cycle analysis and TUNEL staining of cells cultured under hypoxic
conditions. HDMECs were cultured under hypoxic conditions for 0, 6 or 24 hours
as indicated. (A) For cell cycle analysis, DNA was stained using propidium
iodide and analysed by flow cytometry. Hypoxic culture resulted in an
increased number of cells arrested at the G0/G1 phases and an increased level
of DNA fragmentation (blue profile). (B) TUNEL staining was performed on the
same batch of cells used for cell cycle analysis. Apple green staining of
nuclei corresponds to apoptotic cells. Hypoxic culture led to an increased
number of apoptotic cells (original magnification: ×250). All
experiments were performed at least three times and produced similar
results.

Hypoxia alters expression of pro- and anti-apoptotic markers

To investigate the mechanisms by which hypoxia induces cell apoptosis, the
protein expression of the known anti-apoptotic markers Bcl-2,
Bcl-XL, Mcl-1 and the pro-apoptotic markers Bax, caspase-3 and
caspase-8 were quantified in cells under normoxia or hypoxia for 24 hours. As
shown in Fig. 4,
Bcl-XL and Mcl-1 were significantly suppressed, Bax and Caspase-3
were markedly raised and caspase-8 slightly raised in hypoxia. Although Bcl-2
was elevated in hypoxia, the ratio of Bcl-2 to Bax was lowered from 1.36 in
normoxia to 0.79 in hypoxia. These data suggest that hypoxia induces cell
apoptosis through the downregulation of Bcl-XL and Mcl-1,
upregulation of Bax, caspase-3 and caspase-8 and the lowering of the Bcl-2 to
Bax ratio.

Analysis of anti- and pro-apoptotic proteins under hypoxic conditions.
Cells collected after 24 hours of culture under normoxic or hypoxic conditions
were subjected to immunostaining using specific antibodies and FACS analysis.
The ratio of Bcl-2 to Bax dropped from 1.36 in normoxia to 0.79 in hypoxia.
Data were expressed as means±s.e.m. pooled from duplicate samples of
three experiments (*P<0.05 and **P<0.01, student's
t-test).

Suppression of CD105 gene expression using an antisense approach

To investigate the role of CD105 in HDMECs, CD105 gene expression was
specifically inhibited using an antisense approach. There have been numerous
reports in the literature using the same experimental approach, and the method
of transfection is mild and well-tolerated. Thus, examining the effect of
hypoxia on the transfected cells is considered to be appropriate. In
comparison with HUVECs, in which CD105 was significantly reduced using 0.25
nM/ml of AS ODN plus 5.6 μg/ml of lipofectACE (Invitrogen)
(Li et al., 2000a), HDMECs
were less responsive to such a formulation. However, the efficacy was improved
by using an increased concentration of AS ODN (0.5 nM/ml) plus 8 μg/ml of
DMRIE-C (Invitrogen). FACS and immunoblotting analysis revealed a 55-60%
reduction in CD105 protein levels in the AS-ODN-treated cells after 72 hours
of incubation in normoxia and 24 hours exposure to hypoxia, compared with
SC-ODN-treated (0.5 nM/ml plus 8 μg/ml DMRIE-C) or untreated cells under
the same conditions (Fig.
5A,B). To investigate whether CD105 mRNA was altered by ODN
treatment, northern blotting was carried out. As depicted in
Fig. 5C, CD105 mRNA was
decreased by AS but not by the SC ODN, demonstrating that specific degradation
of CD105 mRNA was one of the mechanisms involved in the antisense effect. The
specificity of the antisense effect was further verified by quantifying the
expression of CD31, vWF, TGFβ receptor I and receptor II in the untreated
HDMECs or HDMECs treated with either AS or SC ODN. As determined by FACS and
immunoblotting, these cell membrane proteins were not affected by the ODN
treatment, whereas CD105 protein was significantly reduced by the AS ODN (data
not shown). The selective inhibition of CD105 gene expression by the AS ODN
can also be seen in Fig. 5,
where CD105 but not CD31 protein (A), CD105 but not GAPDH mRNA (C) were
markedly decreased by the AS ODN.

CD105 mRNA and protein expression in ODN-treated and untreated cells.
HDMECs were cultured for 72 hours in normoxia in the presence or absence of
ODN plus DMRIE-C, followed by exposure to hypoxia for 24 hours. CD105 protein
levels were assessed by immunoblotting and FACS, and mRNA levels were assessed
by northern blotting. (A) Immunoblotting revealed that CD105 protein was
reduced by ∼58% in AS-ODN-treated cells (lane 2) compared with
SC-ODN-treated (lane 3) and untreated cells (lane 1). The bar chart indicates
the ratio of CD105 relative to CD31 signal intensity collected from three
experiments (means±s.e.m.). (B) FACS analysis of cell surface CD105
indicated a ∼60% reduction in AS-ODN-treated (blue profile) compared with
SC-ODN-treated (green profile) or untreated (black profile) cells. The red
profile represents a negative control, where mAb E9 was substituted with
pre-immunised mouse serum. The plot is a representative of five similar
experiments. (C) Northern blotting of CD105 and GAPDH mRNA showed that CD105
mRNA was markedly degraded by AS ODN (lane 2) but not by SC ODN (lane 3). Lane
1 is mRNA extracted from untreated cells, and it has no evident alteration
compared with lane 3. The bar chart shows CD105 mRNA, relative to GAPDH mRNA
signal intensity, pooled from three experiments (means±s.e.m.).

Hypoxia stimulates angiogenesis by upregulating expression of angiogenic
factors but causes apoptosis as well. A balance between the two forces
determines the fate of the cell. To elucidate whether upregulation of CD105 in
HDMECs under hypoxic stress is of functional importance, we quantified cell
apoptosis of the AS-ODN-treated, SC-ODN-treated or untreated HDMECs. Exposure
of untreated cells to hypoxic stress for 24 hours elicited a significant cell
apoptosis, 48.5% under hypoxia in contrast to 4.3% under normoxia. Hypoxia
induced a maximal cell apoptosis in the CD105-depressed out of the three
groups of cells cultured in complete growth medium without exogenously added
TGFβ1 or TGFβ3. Thus, the geometric mean fluorescence intensity was
139.1±13.3 in untreated, 150.7±9.0 in SC-ODN-treated and
193.1±10.6 in AS-ODN-treated cells (P<0.05, AS-ODN-treated,
compared with either SC-ODN-treated or untreated cells)
(Fig. 6A). The percentage of
apoptotic cells correlates well with the geometric mean fluorescence
intensity, which was 66.5% in CD105-depressed compared with 48.5% in untreated
or 48.2% in SC-ODN-treated cells (P<0.05)
(Fig. 6B). Administration of
the neutralising antibodies to TGFβ1 and TGFβ3 produced no
significant alteration either in the percentage of apoptotic cells or in their
fluorescence intensity, demonstrating that CD105 is able to act independently
of TGFβ in preventing cell apoptosis under hypoxic stress.

Effect of CD105 antisense ODN on cell apoptosis. Three groups of cells, AS-
or SC-ODN-treated or untreated HDMECs, were exposed to hypoxia and/or
TGF-β1 for 24 hours, TUNEL-stained and analysed by FACS. The fluorescence
intensity (A) and percentage of apoptotic cells (B) were determined. The
apoptotic effect of TGFβ1 was observed to be concentration dependent,
with the maximal effect of TGFβ1 at 10 ng/ml. Data were expressed as
means±s.e.m. collected from six samples of three experiments.
(*P<0.05 and **P<0.01, one-way ANOVA followed by the
Duncan test).

CD105 regulate TGFβ1 signalling in endothelial cells and in CD105
transfectants (Lastres et al.,
1996; Letamendia et al.,
1998; Li et al.,
2000a) but how CD105 and TGFβ1 interact under hypoxic
conditions to control cell apoptosis has not been examined. Therefore we
investigated this issue using the AS-ODN-treated (CD105-deficient) cells, the
SC-ODN-treated and untreated cells. As shown in
Fig. 6, TGFβ1 induced a
marginal increase in apoptotic cells in the SC-ODN-treated and the untreated
cells. In contrast, it caused a considerable increase in apoptotic cells in
the CD105-depressed cells. The fluorescence intensity of CD105-deficient cells
was 193.1±10.6 in the absence of TGFβ1, 229.7±10.7 at 0.1
ng/ml of TGFβ1, and 298.2±11.5 at 10 ng/ml of TGFβ1, in
contrast to 150.7±9.0, 157.9±8.7 and 175.6±10.8,
respectively, in SC-ODN-treated cells (P<0.05 or
P<0.01, significant difference between the two groups at each same
concentration of TGFβ1) (Fig.
6A). This represents 28.1%, 45.5% and 69.8% increases in
fluorescence intensity in the CD105-deficient cells compared with
SC-ODN-treated cells at the same concentrations of TGFβ1. A higher
proportion of apoptotic cells was observed in the CD105-deficient cells
compared with either the SC-ODN-treated or untreated cells at the same
concentration of TGFβ1 (Fig.
6B). The percentage of apoptotic cells increased from
48.2±2.6, 51.3±3.0 and 56.9±3.0 in SC-ODN-treated cells
at 0, 0.1 and 10 ng/ml of TGFβ1 to 66.5±2.3, 75.7±2.6 and
87.4±2.5 in the AS-ODN-treated cells at the same concentrations of
TGFβ1 (P<0.05 or P<0.01, significant difference
between the two groups at each same concentration of TGFβ1). These data
demonstrate that CD105 functions as an anti-apoptotic protein in the presence
of TGFβ1 under hypoxic stress.

Discussion

Hypoxia is a major stimulus of neovascularisation under many conditions,
including tumour angiogenesis, collateral vessel formation in ischaemic
cardiovascular diseases and proliferative retinopathy
(Semenza, 2000;
Marti and Risau, 1999;
Richard et al., 1999;
Smith et al., 1997). CD105 is
upregulated in all these disease states, although its role in angiogenesis is
not fully understood. In this study we demonstrate that hypoxia activates the
CD105 gene promoter, augmenting its mRNA transcription and protein
translation. Most importantly, our data reveal that CD105 exerts an
anti-apoptotic effect in endothelial cells under hypoxic stress, either in the
absence or in the presence of TGFβ1. In view of these observations, it
can be speculated that hypoxia-initiated neovascularisation is mediated, at
least in part, through the stimulation of CD105 expression in ECs.

The notion that CD105 is associated with angiogenesis initially came from
the observation that a mAb to CD105 reacts most strongly with the endothelium
of tumours but only weakly or not at all with normal tissues
(Wang et al., 1993).
Subsequently, numerous studies using mAbs to CD105 on a broad range of tissues
have provided supportive evidence that CD105 is indeed upregulated in many
types of tissues undergoing angiogenesis
(Kumar et al., 1996;
Miller et al., 1999;
Seon and Kumar, 2001;
Wikstrom et al., 2002). The
expression of CD105 in blood vessels of breast and lung cancer tissues was
found to be correlated with poor prognosis
(Kumar et al., 1999;
Tanaka et al., 2001), which
suggests that CD105 promotes tumour progression. A conclusive demonstration of
the crucial role of CD105 in vascular development came from CD105-knockout
mice, which had severe defects in angiogenesis; the homozygotes died in utero
owing to impaired development of vasculature
(Arthur et al., 2000;
Bourdeau et al., 1999;
Li et al., 1999). In line with
these observations, CD105 expression correlates with activation/proliferation
of tumour endothelial cells (Kumar et al.,
1999; Miller et al.,
1999). These findings provide compelling evidence that CD105 is
important for angiogenesis. However, the underlying mechanism of how CD105
promotes angiogenesis is not clear. Existing data support the view that CD105
overexpression in transfectants weakens the effects of TGFβ1 and that
these effects might be cell type dependent
(Lastres et al., 1996;
Letamendia et al., 1998). In
addition, suppression of CD105 in ECs with antisense ODN in combination with
TGFβ1 led to a strong inhibition of angiogenesis
(Li et al., 2000a). These
observations indicate that CD105 modulates TGFβ1 signalling in these
cells and that an adequate level of CD105 in ECs is required for
angiogenesis.

The expression of CD105 is likely to be regulated by factors involved in
angiogenesis and/or vessel remodelling. Here we have demonstrated that CD105
is markedly induced in ECs by exposure to hypoxia. CD105 promoter activity and
mRNA transcription responded rapidly to hypoxia, which suggests that it is
regulated at the transcriptional level. In this regard, a consensus hypoxia
responsive element (HRE) has been recently characterized within the CD105
promoter (Sanchez-Elsner et al.,
2002). The CD105 protein was maintained at high levels until the
end of the experiment. The half-life of CD105 on the cell surface has been
estimated to be 17 hours, measured by metabolic labelling
(Paquet et al., 2001). Thus,
once it is upregulated, CD105 can remain in the EC, exhibiting a prolonged
effect. The relatively low promoter activity and mRNA level after 24 hours of
hypoxic culture is apparently a consequence of hypoxia-induced cell cycle
arrest and apoptosis. This is in line with previous observations that CD105 is
more strongly expressed in activated/mitotic cells than quiescent cells,
suggesting that it is a proliferation-associated gene. These findings also
highlight the potential value of the CD105 promoter for gene therapy in cancer
and ischaemic diseases (Brekken et al.,
2002; Velasco et al.,
2001). Genes driven by the CD105 promoter may be more strongly and
specifically expressed in the oxygen-deprived tissues and exert therapeutic
effects.

Hypoxia is the primary driving force for neovascularisation. The major
angiogenic factors, such as VEGF and bFGF, are upregulated by hypoxia, and
promote angiogenesis and thus improves oxygen supply to the hypoxic tissues
(Mukhopadhyay et al., 1995;
Sakaki et al., 1995;
Schweiki et al., 1992). On the
other hand, persistent hypoxic stress induces EC apoptosis
(Carmeliet et al., 1998;
Hogg et al., 1999). In this
study we have shown that the pro-apoptotic proteins Bax, caspase-3 and
caspase-8 were elevated and that the anti-apoptotic proteins Bcl-XL
and Mcl-1 were significantly decreased under hypoxic stress. Although
anti-apoptotic Bcl-2 was upregulated, the ratio of Bcl-2 to Bax was
considerably lowered under hypoxic stress compared with normoxia. Therefore,
the mechanisms of hypoxia-induced EC apoptosis largely depend on the lowered
Bcl-2 to Bax ratio, Bcl-XL and Mcl-1 and increased expression of
caspase-3 and caspase-8. These findings are in agreement with previous reports
(Khurana et al., 2002;
Taraseviciene-Stewart et al.,
2001; Wang et al.,
2002).

Because CD105 is strongly expressed in the HDMECs under hypoxic conditions,
we adopted an antisense approach to suppress CD105 expression so that its
function could be specifically addressed. The CD105 protein and mRNA levels
were considerably reduced by the antisense ODN but not affected by the control
SC ODN. Using the CD105-deficient cells and the control cells, an important
function of CD105 has been discovered, which is that it acts as an
anti-apoptotic protein in ECs under hypoxic stress. Such an effect was
observed in the absence of TGFβ1 and TGFβ3, indicating that CD105
functions beyond its role as a receptor for TGFβ1 and TGFβ3. In
fact, only approximately 1% of membrane-bound CD105 binds to TGFβ1 and
TGFβ3 (Cheifetz et al.,
1992). The function of the majority of CD105 that does not bind to
TGFβ remains unclear. Our data suggest that the non-TGFβ-binding
CD105 in EC plays a self-protective role against apoptotic factors such as
hypoxia. The protective role of CD105 against apoptosis has been further
confirmed using a completely different system – murine endothelial cells
from CD105 heterozygous knockout mice (J.M.L.-N., C.L., S.K. et al.,
unpublished). The addition of TGFβ1 to control cells under hypoxia
induced a marginal increase in the proportion of apoptotic cells, whereas the
apoptotic action of TGFβ1 was considerably increased in the
CD105-depressed cells, demonstrating that the upregulation of CD105 protects
ECs from the apoptotic action of TGFβ1. In an in vivo environment, where
both hypoxia and TGFβ1 can co-exist, the augmented expression of CD105
may act as an anti-apoptotic force, so as to protect ECs against hypoxia and
against TGFβ1-induced apoptosis.

In conclusion, we have demonstrated that hypoxia activates the CD105
promoter and significantly induces its gene expression in human microvascular
ECs. The upregulated CD105 exhibits a role in self-protection against hypoxia
and TGFβ1-induced cell apoptosis, resulting in an enhanced survival
ability of EC under hypoxic stress. These findings may lead us to a better
understanding of the functions of CD105 in angiogenesis and of the
pathogenesis of other CD105-related vascular disorders.

Andreas Villunger and colleagues discuss the biology of the PIDDosome multiprotein complex and recent advances that link PIDDosome-dependent CASP2 activation to p53 activation in response to extra centrosomes.

Centrosomes and cilia are essential structures for many functions in development and disease. Sascha Werner, Ana Pimenta-Marques and Mónica Bettencourt-Dias review how their structure and functions are maintained.

Marian Blanca Ramírez from the CSIC in Spain has been studying the effects of LRRK2, a protein associated with Parkinson’s disease, on cell motility. A Travelling Fellowship from Journal of Cell Science allowed her to spend time in Prof Maddy Parson’s lab at King’s College London, learning new cell migration assays and analysing fibroblasts cultured from individuals with Parkinson’s. Read more on her story here.

Where could your research take you? The deadline to apply for the current round of Travelling Fellowships is 30 Nov 2017. Apply now!